Heparnoid (Heparin and Heparan sulfate) is a well-known regulatory mediator in numerous important biological processes. Heparnoid and its derivative, low-molecular weight heparin (LMWH), have been used as clinical anticoagulant drugs during surgery and kidney dialysis. For example, Fondaparinux sodium (CAS 114870-03-0) is a member of oligosaccharides/heparins with a chemical name of O-[2-Deoxy-6-O-sulfo-2-( sulfoamino)-alpha-D-glucopyranosyl]-(1-4)-O-(beta-D-glucopyranurosonyl)-(1-4)-O-[2-deoxy-3,6-di-O-sulfo-2-(sulfoamino)-alpha-D-glucopyranosyl]-(1-4)-O-(2-O-sulfo-alpha-L-idopyranurosonyl)-(1-4)-O-[2-deoxy-1-O-methyl-6-O-sulfo-2-(sulfoamino)-alpha-D-glucopyranoside] decasodium salt, and was developed by Choay, S. A. (see U.S. Pat. No. 4,818,816). The compound is a synthetic pentasaccharide Factor Xa inhibitor which is used as an anticoagulant drug for the prevention of deep vein thrombosis in patients who have had orthopedic surgery as well as for the treatment of deep vein thrombosis and pulmonary embolism. Fondaparinux sodium was approved by the United States Food and Drug Administration in 2001, marketed under the trade name Arixtra™. Fondaparinux sodium is administrated subcutaneously.
Analytical methods for heparin and heparan sulfate, traditionally involved reverse phase chromatographic and mass spectrometric (MS) techniques, but have limitations due to the high polarity, structural diversity, and sulfate lability of heparan sulfate. For instance, the quantitation of synthetic poly-sulfated oligosaccharides using MS is restricted because the ionization of poly-sulfated oligosaccharides tends to form various types of fragments and metal cation-coupled adducts with loss of sulfate groups. This leads to greater spectral complexity and signal splitting. In addition, it is difficult to demonstrate the degree of loss of sulfate groups during the analysis because it depends on the concentration and charge state of sulfated oligosaccharides. Improved analytical methods for poly-sulfated oligosaccharides have been the target of a number of research groups.
Catalin et al. (Anal. Chem. 2009, 81, 3485) and Tatiana et al. (Anal. Chem. 2006, 78, 1774) have each described the characterization of poly-sulfated oligosaccharides by using electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser desorption and ionization mass spectrometry (MALDI-MS). However, the current methods using the coupling of liquid chromatograph (LC) with mass spectrometry does not provide on-line in-process resolution/separation of peaks and hence the identification of the structure related impurities and/or quantitation of the poly-sulfated oligosaccharides cannot be established during the production of synthetic poly-sulfated oligosaccharides.
Imanari et al. (J. Chromatogr., A 1996,720, 275.) and Rice et al. (J. Anal. Biochem. 1985, 150, 325.) illustrated the analytical method of poly-sulfated oligosaccharides by strong anion exchange chromatography (SAX). This method appears to separate highly sulfated oligosaccharides via the difference in charge density, but it remains difficult to directly couple SAX with a detection method like MS due to the use of nonvolatile salt in the mobile phase composition.
Still other problems associated with analytical methods for poly-sulfated oligosaccharides are due to the non-chromophore characteristics (very low UV absorption) of poly-sulfated oligosaccharides, which can restrict the use of traditional UV detectors. The other universal detectors such as refractive index (RI) and evaporative light scattering (ELSD) also lack enough detecting sensitivity for poly-sulfate oligosaccharides.
Although some methods of detection of poly-sulfated oligosaccharides have been disclosed, a number of limitations remain. Thus, there is a continuing need for improved methods for the separation, quantitation and mass identification of poly-sulfated oligosaccharides. The stable, sensitive and in-process control (IPC) methods disclosed herein address this need and other needs.
BRIEF SUMMARY OF THE INVENTION
Provided herein is a method for detecting poly-sulfated oilgosaccharides, using a hydrophilic interaction ultra-performance liquid chromatography (HILIC-UPLC) coupled with a charged aerosol detector (CAD) or a mass spectrometer (MS). The methods provided herein allow for improved peak resolution thereby allowing for subsequent quantitation of poly-sulfated oligosaccharides and/or impurities in the sample.
The use of HILIC overcomes the challenge of retaining and separating extremely polar oligosaccharides. The retention mechanism for HILIC is very intricate and is a multi-modal combination of liquid-liquid partitioning, adsorption, ionic interaction and hydrophobic interaction. Therefore, HILIC, in comparison to reverse phase liquid chromatography (RPLC), provides unique selectivity and retention characteristics.
As provided herein, the stationary phase used in HILIC column is, in one group of embodiments, an amide-bonded stationary phase.
In another embodiment, the mobile phase used in HILIC column comprises a salt. In one group of embodiments, the salt is ammonium formate. The use of ammonium formate, in comparison to pyridinium formate and ammonium acetate, provides better performance for retention, selectivity and low noise level baseline.
In some embodiments, the concentration of the salt is higher than 50 mM. In some selected embodiments, the concentration is higher than 100 mM. Typically, the molar strength of the salt additive in the mobile phase composition can have a significant impact on chromatographic retention, selectivity and sensitivity. As the molarity of the salt additive increases, the ionic strength of the mobile phase and the solute is overpowered by the liquid-liquid partitioning interaction which dominates the retention mechanism rather than the ion exchange effect. However, it has now been discovered that in the case of acidic analytes, such as poly-sulfated oligosaccharides, retention is enhanced as the molarity of the salt additive increases. In particular, the resolution of peaks is further improved as the salt concentration is increased from 50 mM to about 200 mM.
In one group of embodiments, the solvent of the mobile phase used in the HILIC column is acetonitrile.
In some embodiments, the detector used for the quantitation of poly-sulfated oligosaccharides is a charged aerosol detector (CAD). During the analysis using CAD, aerosol particles are charged with an ionized gas (typically nitrogen). After the removal of high-mobility particles (mainly excess N2 ions), the aerosol particles are then electrically measured. Most importantly, the method has been demonstrated to provide a uniform response for nonvolatile analytes independent of their nature. Thus, the combination of (1) a separation technique utilizing HILIC, or HILIC-UPLC, and (2) a detection technique such as MS or CAD allows for detection, identification and/or quantification of poly-sulfated thereby providing an effective way for analysis of synthetic poly-sulfated oligosaccharides.
In accordance with one selected embodiment of the present invention, the poly-sulfated oilgosaccharide detected and/or quantitated by the methods described herein is Fondaparinux sodium.